It is the long-term goal of this research project to identify molecular mechanisms used by the retinal pigment epithelium (RPE) to phagocytose photoreceptor outer segment fragments (POS) shed in a circadian rhythm in the mammalian eye and to understand how specific changes in these mechanisms due to aging and mutations cause retinal degeneration. Digesting several thousand POS disks every day for life, post-mitotic RPE cells are the most active phagocytes known. Abnormalities in RPE phagocytosis directly contribute to incurable human blinding diseases affecting millions, including age-related macular degeneration and retinitis pigmentosa. Identifying the contributions of the RPE to the fundamental retinal process of outer segment renewal is thus directly relevant to our understanding of human retinal disease. Recently, we developed and explored novel experimental approaches that allow for the first time to directly and quantitatively determine how genetic mutations and age-related changes alter activity of phagocytic proteins by the RPE in vivo and in culture. Shed POS require ligation of aV5 receptors of the RPE by their retinal ligand MFG-E8 to stimulate a cytoplasmic signaling pathway in RPE cells via focal adhesion kinase towards the tyrosine kinase receptor MerTK that is indispensable for efficient POS engulfment. Synchronized POS clearance via aV5 receptors is essential for long-term retinal health: lack of phagocytic rhythm in aging aV5 - deficient mice causes excessive accumulation of pro-oxidant RPE lipofuscin and loss of vision. In aim 1 we propose to identify the missing molecular links between aV5 and MerTK surface receptors and unravel the role of Src kinase in RPE phagocytosis. A novel substrate screen will identify direct Src substrates specific to POS phagocytosis and determine if Src activity is sufficient to promote MerTK activation and POS engulfment. Our preliminary data show that RPE cells following an initial burst of POS binding retain ample surface aV5 and MerTK receptors but fail to promote prolonged POS binding or engulfment signaling. In aims 2 and 3 we therefore propose to identify molecular mechanisms used by RPE cells to actively and specifically inhibit aV5 and MerTK functions following completion of diurnal POS clearance. We will test the hypothesis that RPE cells terminate POS phagocytosis by specific proteolysis of integrin associated proteins that severs anchorage of aV5 receptors to the actin cytoskeleton, and by stimulating specific protein tyrosine phosphatases that inactivate MerTK. Mechanisms that prevent untimely or excess RPE phagocytic activity have long been assumed to exist but to our knowledge never been studied. We propose that such inhibitory mechanisms may ensure the healthy balance of POS shedding and engulfment in the retina. We have procured appropriate in vivo models, specific activity assays and detection reagents for their identification